Guanine nucleotide exchange factors, or GEFs, activate GTPases to control a broad array of cellular behaviors. They integrate diverse cell stimuli and coordinate the localized dynamics of multiple subcellular systems, including the cytoskeleton, adhesion complexes, transcription machinery, and trafficking compartments. GEFs are capable of such diverse roles because they have complex overlapping specificities for downstream GTPases, with interactions controlled by the shifting localization of both the GEFs and GTPases, and by GEF activation kinetics. To fully understand GEF function and specificity, one must study the spatio-temporal dynamics of GEF activation, and transient GEF interactions with specific targets, in living cells. Here we will make this possible by generating the first fluorescent biosensors of GEF proteins. We are purposefully targeting GEFs representative of different structural classes, to develop generalizable biosensor designs that can open the door to biosensors for many different GEFs. We will explore new biosensor technologies, including genetically encoded biosensors that report activation of endogenous GEFs, and complementary biosensors that can report either the overall activation of a GEF or activation only by specific stimuli. The complementary biosensors will enable us to dissect out the contributions of different upstream pathways activating the same GEF for different purposes in LPA-induced cell motility. Each project in the PPG will use the biosensors, together with computational and modeling approaches developed by Project 2 (Danuser/Hahn), to study the coordinated activation of Dbl family GEFs and Rho family GTPases in different biological contexts.